The present invention relates to methods and apparatuses for flow-through capture and optional recovery of nucleic acids.
Nucleic acid hybridization, i.e., the ability of nucleic acid strands of complementary sequence to form duplexed hybrids, is one of the most powerful analytical techniques in the biological sciences. One of the most widely used hybridization techniques today is the xe2x80x9cSouthern blotxe2x80x9d method discovered by Southern (Southern, 1975, J. Mol. Biol. 98:503-507). In this method, a target denatured DNA is immobilized on a filter or membrane, such as a nitrocellulose or nylon membrane. The membrane is then incubated in a buffer solution which contains a labeled oligonucleotide probe complementary to a region of the immobilized target DNA under conditions wherein the target and probe hybridize. Following wash steps, the presence or absence of hybridization is determined by detecting the label, with a positive detection indicating the presence of hybridization. The above method has also been used with immobilized RNA targets. When used with RNA the method is called xe2x80x9cNorthern blotting.xe2x80x9d
While powerful methods, Southern and Northern blotting suffer from several drawbacks. First, the methods cannot be used to study multiple sequences simultaneously within the same membrane in a single run, i.e., without the time-consuming procedures of repeat hybridization by different probes. Second, available membranes are generally unable to provide high immobilization efficiencies for target nucleic acid fragments containing fewer than 100 bp. Third, the hybridization kinetics are slow; oftentimes several hours or even several days are required for the probe and target to form a hybridized complex. Lastly, the Southern and Northern techniques suffer from the drawback that the target nucleic acid cannot be efficiently eluded from the membranes for subsequent use.
The slow hybridization kinetics observed with the Southern and Northern methods are thought to be caused by three main factors. First, since the whole membrane must be covered with hybridization solution, the concentration of probe available for hybridizing to the immobilized target DNA or RNA is extremely low. Since hybridization kinetics are governed by a bimolecular collision process, the dilute probe concentration has an enormous effect on the rate by which the probe xe2x80x9cfindsxe2x80x9d and hybridizes to the target DNA or RNA. Second, the majority of the probe solution does not contact the membrane during the incubation process. This lowers the effective probe concentration even further, and also increases the likelihood that, if the target was initially double-stranded, the target strands will re-anneal at a faster rate than hybridization will occur. Third, a large proportion of the target nucleic acid is immobilized within the interior pores of the membrane, and is therefore inaccessible for hybridization. Thus, the hybridization kinetics are slowed even further by the probe having to diffuse into the pores of the membrane.
Recently, it has been postulated that hybridization can be used to sequence DNA or RNA. The sequencing by hybridization method (xe2x80x9cSBHxe2x80x9d), first described by Lysov et al. utilizes a set of short oligonucleotide probes of defined sequence to search for complementary sequences on a longer strand of target DNA or RNA. The hybridization pattern is then used to reconstruct the sequence of the target DNA or RNA (Lysov et al., 1988, Dokl. Acad. Nauk SSSR 303:1508-1511; see also, Bains and Smith, 1988, J. Theor. Biol. 135:303-307; Drmanac et al., 1989, Genomics 4:114-128; Strezoska et al., 1991, Proc. Natl. Acad. Sci. USA 88:10089-10093; Drmanac et al., 1993, Science 260:1649-1652).
Since the emergence of SBH, many new techniques for fabricating immobilized sets of probes have emerged. For example, Southern et al. constructed an array of 256 octanucleotides covalently attached to a glass plate using a solution-channeling device to direct the oligonucleotide probe synthesis (Southern et al., 1992, Genomics 13:1008-1017). Because the identity of the probe at each site is known, the entire array can be simultaneously hybridized with the target nucleic acid in a single assay; the hybridization pattern directly reveals the identities of all complementary probes.
In a similar vein, Pease et al. describe the use of photoprotected nucleoside phosphoramidites and light to direct the synthesis of a miniaturized array of 256 octanucleotides on a glass substrate in a spatially-addressable fashion (Pease et al., 1994, Proc. Natl. Acad. Sci. USA 91:5022-5026). The resulting miniaturized array measured 1.28xc3x971.28 cm and took only 16 reaction cycles and 4 hours to synthesize. Like the array of Southern, the miniaturized array can be simultaneously hybridized with the target nucleic acid to reveal the identities of all complementary probes.
Dubiley et al. describe the use of oligonucleotide microchips that have been manufactured by immobilizing presynthesized oligonucleotides within polyacrylamide gel pads arranged on the surface of a microscope slide (Dubiley et al., 1997, Nucl. Acids Res. 25(12):2259-2265). The microchips have been applied to sequence analysis (Yershov et al., 1996, Proc. Natl. Acad. Sci. USA 93:4913-4918), mutation analysis (Drobyshev et al., 1997, Gene 188:45-52) and identification of microorganisms.
Hybridization with the above-described probe arrays provides at least two advantages over the Southern and Northern blotting techniques. First, since each probe is attached to a discrete site on the substrate, the target DNA or RNA can be assayed for its ability to form hybrids with a plurality of probes in a single experiment. Second, the hybridized complexes can be readily dissociated and the target nucleic acid recovered for subsequent use. However, since these methods also rely on immersion hybridization techniques, i.e., the entire substrate must be immersed in hybridization solution containing the target nucleic acid, the kinetics of hybridization are slow. Depending on the concentration and length of the target nucleic acid, the formation of hybridized complexes can take on the order of hours or even days. Moreover, the methods require a large volume of hybridization buffer, and hence quite a large quantity of target nucleic acid.
Due to the ability of nucleic acids to form duplexes with a high degree of specificity, hybridization has also been used to capture a target nucleic acid from a sample. Such methods can be used to determine whether the sample contains the target nucleic acid, to quantify the amount of target nucleic acid in the sample, or to isolate the target nucleic acid from a mixture of related or unrelated nucleic acids. To this end, capture polynucleotides capable of hybridizing to a target nucleic acid of interest have been immobilized on a variety of substrates and supports for use in capture assays.
For example, capture polynucleotides have been immobilized within the wells of standard 96-well microtiter plates (Rasmussen, et al., 1991, Anal. Chem. 198:138-142), activated dextran (Siddell, 1978, Eur. J. Biochem. 92:621-629), diazotized cellulose supports (Bunneman et al., 1982, Nucl. Acids Res. 10:7163-7180; Noyes and Stark, 1975, Cell 5:301-310) polystyrene matrices (Wolf et al., 1987, Nucl. Acids Res. 15:2911-2926) and glass (Maskos and Southern, 1992, Nucl. Acids Res. 20:261-266), to name a few. However, these systems suffer from very poor diffusion characteristics, leading to slow, inefficient hybridization.
In part to overcome the slow hybridization kinetics of available immobilization supports, the art has also attempted to immobilize capture polynucleotide on beads, including submicron latex particles (Wolf et al., 1987, supra), avidin-coated polystyrene beads (Urdea et al., 1987, Gene 61:253-264) and magnetic beads (Jakobson et al., 1990, Nucl. Acids Res. 18:3669). However, the beads are difficult to manipulate, particularly magnetic beads, which require elaborate isolation stations to retain the beads and precise liquid handling to avoid removal of the beads from solution.
While the art has attempted to overcome the manipulation problems of non-magnetic beads by packing the beads into columns, such columns are not easily assembled without the aid of unique frits or membranes to retain the beads. Moreover, while columns packed with such beads exhibit more favorable hybridization kinetics than the xe2x80x9cimmersionxe2x80x9d techniques described above, the kinetics are nowhere near optimal, and the columns exhibit enormous back-pressure when hybridization solution is flowed through, which is most likely caused by close-packing of the beads.
Recently, flow-through hybridization devices designed to overcome the adverse kinetics of immersion hybridization methodologies have been designed. The devices utilize a capture polynucleotide immobilized on a membrane. The hybridization reaction takes place as fluids flow through the membrane. In one such device, the capture polynucleotide was immobilized on a nylon or nitrocellulose membrane using UV irradiation or through the use of a specific binding partner, such as biotin (EP 0 605 828 A1). In another such device, the capture polynucleotide, modified at its 5xe2x80x2-terminus with an amino group, was covalently attached to the carboxyl groups of a Biodyne(trademark) C membrane (U.S. Pat. No. 5,741,647). Upon flowing a liquid sample containing a target nucleic acid through the membrane, rapid hybridization (i.e., on the order of minutes) was observed with each device.
Yet, these flow-through devices are not without drawbacks. The membranes used have extremely small pores (about 0.1-0.45 xcexcm) such that even moderate flow-through rates cause significant back-pressure in the device. Moreover, the small pore size leads to clogging, which further increases the back-pressure of the device and may even lead to tearing of the membrane.
The clogging problem seriously limits the utility of the devices. For example, they cannot be used to capture nucleic acids from samples that contain cellular or large molecule contaminants such as, for example, proteins, carbohydrates, RNAs, DNA sequencing templates, etc., as these contaminants clog the pores of the membranes.
Lastly, while the devices can be used for nucleic acid capture, recovery efficiencies are too low to be useful for applications requiring post-capture recovery, such as recovery of PCR fragments, RNAs, restriction-digested DNA fragments, etc.
As the above discussion attests, there remains a need in the art for easy-to-use substrates which provide for rapid, efficient and highly specific capture of target nucleic acids, and which further permit recovery of the captured nucleic acid for subsequent use. Accordingly, these are objects of the present invention.
These and other objects are furnished by the present invention, which in one aspect provides an apparatus for rapidly, efficiently and specifically capturing, and optionally recovering, nucleic acids. In its broadest sense, the apparatus of the invention comprises a porous substrate which has a capture polynucleotide immobilized thereon, typically by way of a covalent bond between the 5xe2x80x2- or 3xe2x80x2-terminus of the capture polynucleotide and a reactive group on the porous substrate, either with or without the aid of one or more linkers. To permit high flow-through rates, the porous substrate generally has an average pore size of about 1 xcexcm to 250 xcexcm and a porosity of about 25% to about 80%. The density or surface concentration of immobilized capture polynucleotide is preferably in the range of about 2xc3x9710xe2x88x9219 to 2xc3x9710xe2x88x9215 nmole/nm2 The porous substrate is optionally disposed within a housing, such as a chromatography column, spin column, syringe-barrel, pipette, pipette tip, 96 or 384-well plate, microchannels, capillaries, etc., which aids the flow of liquids through the porous substrate.
In use, a sample containing or suspected of containing a target nucleic acid capable of hybridizing to the capture polynucleotide is flowed through the porous substrate under conditions wherein the target nucleic acid and capture polynucleotide hybridize. Quite surprisingly, it has been found that the sample need only contact the porous substrate for less than a minute, typically on the order of only 3 to 15 sec., for efficient hybridization to occur, although longer contact times may be used. Following optional wash steps, the presence or absence of hybridization can then be determined.
In one embodiment of the invention, the presence of hybridization is determined by analyzing the porous substrate for the presence of a hybridization-induced detectable signal, such as, for example, fluorescence or chemiluminescence. In another embodiment of the invention, the presence of hybridization is determined by dissociating the hybridized complex, recovering the dissociated target nucleic acid and detecting the presence of the dissociated target nucleic acid.
The flow-through hybridization apparatus of the invention can be used in a wide variety of applications where knowledge about the presence or quantity of a particular target nucleic acid in a sample is desired, or where the capture of a particular target nucleic acid is desired. Thus, in another aspect, the present invention provides methods of using the flow-through hybridization apparatus to determine whether a target nucleic acid is present in a sample. Generally, the method comprises the steps of:
(a) providing a porous substrate having a capture polynucleotide capable of hybridizing to a target nucleic acid immobilized thereon;
(b) flowing a sample suspected of containing the target nucleic acid through the porous substrate under conditions wherein the target nucleic acid and capture polynucleotide hybridize; and
(c) detecting the presence of hybrids, wherein a positive detection indicates the sample contains the target nucleic acid.
In another aspect, the present invention provides a method of capturing a target nucleic acid present in a sample. In general, the method comprises the steps of:
(a) providing a porous substrate having a capture polynucleotide capable of hybridizing to the target nucleic acid immobilized thereon; and
(b) flowing a sample containing or suspected of containing the target nucleic acid through the porous substrate under conditions wherein the target nucleic acid and capture polynucleotide hybridize, thereby capturing the target nucleic acid. Following capture, the target nucleic acid can be optionally recovered by dissociating the hybridized complex and used in subsequent methods, such as for example, sequencing.
In a final aspect, the invention provides kits for a variety of capture applications. Generally, the kits comprise a three dimensional porous substrate of the invention having immobilized thereon a capture polynucleotide capable of hybridizing with a target nucleic acid of interest and one or more other reagents or components useful for performing a particular assay. Alternatively, the kits can comprise a three dimensional porous substrate activated with a reactive functional group and a capture polynucleotide modified with a group capable of forming a covalent linkage with the activated porous substrate, or means for synthesizing a capture polynucleotide on the activated substrate, such as nucleoside phosphoramidites and/or other DNA or RNA synthesis reactants or reagents. Optional components that can be included with the kits include housings in which the substrates can be disposed, sequencing templates and dideoxynucleotide reagents and enzymes for generating sequencing ladders from the target nucleic acid, polymerases and primers for amplifying the target nucleic acid, linkers for spacing the target nucleic acid from the porous substrate and buffers and reagents useful for sequencing, amplification and/or other nucleic acid applications.